Thrombin is a serine protease that plays opposing functional roles in blood coagulation: it is both a procoagulant that converts fibrinogen into an insoluble fibrin clot, and an anticoagulant that activates protein C, and thrombin also has an extracellular protease-activated receptor (PAR) activation function (Di Cera, “Thrombin”, Mol Aspects Med 2008 August; 29(4): 203-254, doi: 10.1016/j.mam.2008.01.001, Epub 2008 Feb. 1). Wild-type thrombin expressed in mammalian cells is often used for its procoagulant properties, particularly to address the problem of surgical bleeding. Thrombin variants engineered for optimal activity toward protein C and minimal activity toward fibrinogen have shown remarkable anticoagulant and antithrombotic properties of therapeutic interest, both in vitro and in vivo (Cantwell and Di Cera, “Rational design of a potent anticoagulant thrombin”, J Biol Chem 2000 Dec. 22; 275(51): 39827-39830; Epub 2000 Nov. 1; Gruber et al., “The thrombin mutant W215A/E217A shows safe and potent anticoagulant and antithrombotic effects in vivo”, J Biol Chem 2002 Aug. 2; 277(31): 27581-27584; Epub 2002 Jun. 17; U.S. Pat. Nos. 6,706,512; 7,223,583). The antithrombotic effect of anticoagulant thrombin variants in non-human primates is more efficacious than the direct administration of activated protein C, and is safer to use than the administration of low molecular weight heparins (Gruber et al., “Relative antithrombotic and antihemostatic effects of protein C activator versus low-molecular-weight heparin in primates”, Blood 2007 May 1; 109(9): 3733-3740; Epub 2007 Jan. 16).
The production of thrombin in vivo proceeds through a series of intermediate forms, including prothrombin, prethrombin-1, and prethrombin-2. Each step of this process involves a highly regulated cleavage of the precursor form of thrombin, until mature thrombin is produced (DiBella et al., “Expression and folding of recombinant bovine prethrombin-2 and its activation to thrombin”, J Biol Chem 1995 Jan. 6; 270(1): 163-169). Mature thrombin is formed of two polypeptide chains, the A chain and the B chain, with a disulfide bond between the A and the B chain, and can be produced by cleavage of prethrombin-2 at a specific site.
Thrombin, including anticoagulant thrombin variants, has been expressed in cultured mammalian host cells such as BHK21 cells (Guinto et al., “Unexpected crucial role of residue 225 in serine proteases”, Proc Natl Acad Sci USA 1999 Mar. 2; 96(5): 1852-1857; Cantwell and Di Cera 2000, supra), and in E. coli (US Patent Application Publication 2012/0164129 A1). Thrombin prepared in this way is typically expressed as prethrombin-2, which needs to be converted to mature thrombin (or “activated”) by treatment with an enzyme, for example by treatment with prothrombinase complex which contains activated factor X, or immobilized ecarin, an enzyme purified from snake venom, or other activators.
Therefore, there exists a need for alternative methods of producing mature recombinant thrombin that do not require additional expensive or inconvenient steps such as refolding reactions (if expressed in bacteria as inclusion bodies), or purification of a thrombin precursor from the media, followed by activation of the precursor to thrombin with an activator.